Production of bio-based materials using photobioreactors with binary cultures

ABSTRACT

A method, device and system for producing preselected products, (either finished products or preselected intermediary products) from biobased precursors or CO 2  and/or bicarbonate. The principal features of the present invention include a method wherein a binary culture is incubated with a biobased precursor in a closed system to transform at least a portion of the biobased precursor to a preselected product. The present invention provides a method of cultivation that does not need sparging of a closed bioreactor to remove or add a gaseous byproduct or nutrient from a liquid medium. This improvement leads to significant savings in energy consumption and allows for the design of photobioreactors of any desired shape. The present invention also allows for the use of a variety of types of waste materials to be used as the organic starting material.

PRIORITY CLAIM

This application is a continuation in part of application Ser. No.12/555,631 filed Sep. 8, 2009 which claims priority from provisionalpatent application No. 61/095,413 filed Sep. 9, 2008 and 61/099,380filed Sep. 23, 2008 the contents of each are herein incorporated byreference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY-SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under ContractDE-AC0576RLO1830 awarded by the U.S. Department of Energy. TheGovernment has certain rights in the invention.

BACKGROUND

Rising fuel prices and global climate change concerns have revived theinterest in renewable sources of energy. Using solar energy to growphotosynthetic microorganisms is one of the most attractive ways toproduce transportation fuels. Successful implementation of biodiesel viaseed crops is one example of employing plant-based photosynthesis forfuel production. However, recent assessments of crop-based fuel economyshowed that it can lead to food stock deficiency and drive lifecycleemissions of greenhouse gases up through increased land usage.Utilization of photosynthetic microorganisms for primary biomassproduction has many advantages over growing crops. In particular, aridregions of the western U.S., for example, could be used for large-scaleproduction excluding the competition with food-producing agriculture.

Cultivation of photoautotrophic microorganisms for metabolite and/orbiomass production can be accomplished in various types of cultivationsystems including open ponds and enclosed bioreactors. Each system hasvarious advantages and limitations. Open ponds, for example, aredesigned to utilize natural sunlight while most of the enclosedbioreactor systems do require artificial illumination which results inadditional energy expenditures. Open pond systems, however, are moreprone to fouling by external contamination and are not suited to growgenetically modified organisms. In contrast, enclosed bioreactorsprovide highly controlled conditions, protection against externalcontamination, and higher growth rates and biomass/products yields whileallowing use of genetically modified strains. Cultivation ofphotosynthetic organisms is also associated with several generalproblems which arise from the necessity to deliver CO₂ into liquidmedium and remove excess O₂ produced as a result of photosynthesis inorder to maintain desired growth conditions. The current practice is tocontinuously or periodically purge the system which adds significantlyto the operating costs and results in frequent changes of cultivationconditions and reduction in efficiency. Removal of O₂ by most otherknown methods such as by chemical catalysis is typically prohibitivelycostly. What is needed therefore is a solution that enables continuousoperation under controlled conditions such as within an enclosedbioreactor without the need for venting as is required by the prior art.The present invention meets this need.

SUMMARY

The present invention is a method, device and system for producingpreselected products, (either finished products or preselectedintermediary products) from biobased precursors. The principal featuresof the present invention include a method wherein a culture consistingof two microorganisms (binary culture), is incubated with a biobasedprecursor in a closed system to transform at least a portion of thebiobased precursor to a preselected product. However, a variety of otheraspects including the particular systems and devices which enable themethod of the present invention to be performed are also described anddisclosed herein.

In some embodiments the binary culture includes at least one oxygenicphotoautotroph and at least one aerobic or facultative anaerobicchemoheterotroph. In other embodiments the pairings may include any setof organisms appropriately combined so that the rates of the growth ofthe two strains of organisms are maintained in an appropriate balanceand the internal environment is maintained in a desired condition. Thisincludes but is not limited to pairings of various strains of bacteria,algae, fungi and plant species and combinations thereof. Examples couldinclude but are not limited to pairings of facultative aerobic andanaerobic organisms to produce a self-sustaining oxygen/carbon dioxidebalance, as well as other combinations wherein the two organisms producedesired or required nutrients or growth enhancing materials.

In some embodiments the binary cultures are incubated in a closedchamber and artificially illuminated by variously placed light emittingdiodes which are controlled by a control mechanism based uponinteraction with a plurality of sensors within the chamber. In otherapplications the present invention may utilize natural sunlight orambient light or combinations of ambient and directed light in order toobtain a desired effect. In one embodiment of the invention, the systemis completely artificially lighted and the emissions from the lightemitting sources are coordinated by interaction between a these lightemitting sources preferably (light emitting diodes) having selectedcharacteristics such as wavelength, frequency, intensity or otherfeatures and at least one sensor (preferably a plurality of sensors areutilized) that is located within the chamber by a computer program. Inaddition to the maintaining of optimal lighting conditions within thechamber, the temperature of the internal contents of the chamber canalso be variously monitored and controlled to provide an optimaltemperature for growth. A heat sink enables excess or unwanted heat tobe removed from the chamber, while the light inside the chambertypically provides the heating means for raising the internaltemperature to a desired value. In one embodiment of the invention, theheat sink is an integrated part of the cylindrical chamber, in otherembodiments additional heat sinks may be attached to the cylinder inorder to obtain a desired result. Preferably a cylinder having a heightto width aspect ratio of at least 2 is considered optimal in suchcircumstances.

A non-aerated photobioreactor for obtaining biotechnology products usingCO₂ and light together with a binary culture made up of a combination ofa phototrophic organism and a heterotrophic organism saidphotobioreactor having a chamber with a height to diameter rationgreater than two and lit by an artificial lighting system comprised oflight emitting diodes (LEDs) arranged in a spaced configuration.

In one embodiment of the invention the binary culture includes at leastone photoautotroph and at least one chemoheterotroph. In one examplethis may be selected from any of a variety species including but notlimited to Shewanella species, Cyanothece species, Synechococcus speciesand other species appropriate for the particular necessities of a user.One of the advantages that the present invention provides is that unlikepreviously used methods for O₂ removal/CO₂ delivery, this method ofcultivation does not need purging of a closed bioreactor with a definedgas phase. In contrast to most prior art practices for O₂ removal/CO₂delivery required for microalgae cultivation, the proposed approachaccomplishes these tasks simultaneously by inclusion of a compatibleheterotrophic microorganism. This method of cultivation does not requiresparging of a closed bioreactor with air or other gas mixture/vigorousmixing to deliver CO₂/remove produced O₂ from liquid medium. Thisimprovement leads to significant savings in energy consumption andallows for the design of photobioreactors of any desired shape to ensureoptimal photoautotrophic culture illumination and space usage which willultimately result in designing more efficient processes with substantialincreases in biomass production and/or product generation. Additionally,use of highly reduced organic compounds will help to consume externallyadded CO₂ or a salt of carbonic acid without necessity to remove O₂.

The present invention also allows for the use of a variety of types ofwaste materials to be used as the organic starting material. Forexample, biosludge produced from sewage water treatment plants orglycerol, a major dead-end by-product in biodiesel production can beutilized. In addition the present invention can be utilized for theproduction of microalgae biomass as feedstock for high-quality biofuels(biodiesel and biocrude) that require a minimum of post-productionprocessing. The present invention also enables the design oflight-driven processes for bio-H₂ production and the production oforganic fertilizers, animal feed, and other commodities including butnot limited to vitamins, amino acids, antibiotics, or enzymes. Thepresent invention solves the problems associated with the prior art byutilizing binary cultures of paired organism to produce self-sustaininginterdependencies that foster the continued growth and development ofthe organism producing the desired biomass material within the closedsystem wherein the growth environment can be carefully monitored andmaintained. Additionally, more than two cultures may be grown togetheras necessary. Such an approach provides a cost-efficient way toeliminate problems associated with the prior art methodologies.

In one embodiment of the invention the paired binary cultures areconfigured to provide CO₂ delivery and O₂ removal while creatinghigh-value products by utilizing sun light, artificial light or theircombination and organic matter (waste or renewables). In one exemplaryembodiment a photoautotrophic organism such as a microalga or acyanobacterium is paired with an aerobic or facultative anaerobicheterotrophic bacterium. The phototrophic oxygenic microorganisms canproduce biofuels at a much higher productivity than land plants and canbe cultivated in aquatic environments, including seawater, so as to notcompete for resources with conventional agriculture. In most prior artsystems high costs associated with increasing the mass transfer andby-product (O₂) removal limit its use, however in the present embodimentthese materials are consumed by an aerobic or a facultative anaerobicheterotrophic bacteria and the desired level of homeostasis within theclosed chamber is maintained. This method can be employed by a varietyof systems wherein binary cultures of paired organisms cooperativelyco-exist to maintain a desired growth environment depending upon theparticular needs and necessities of a user.

The purpose of the foregoing abstract is to enable the United StatesPatent and Trademark Office and the public generally, especially thescientists, engineers, and practitioners in the art who are not familiarwith patent or legal terms or phraseology, to determine quickly from acursory inspection the nature and essence of the technical disclosure ofthe application. The abstract is neither intended to define theinvention of the application, which is measured by the claims, nor is itintended to be limiting as to the scope of the invention in any way.

Various advantages and novel features of the present invention aredescribed herein and will become further readily apparent to thoseskilled in this art from the following detailed description. In thepreceding and following descriptions We have shown and described onlythe preferred embodiment of the invention, by way of illustration of thebest mode contemplated for carrying out the invention. As will berealized, the invention is capable of modification in various respectswithout departing from the invention. Accordingly, the drawings anddescription of the preferred embodiment set forth hereafter are to beregarded as illustrative in nature, and not as restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of the system and process of the presentinvention.

FIG. 2 a is an exemplary view of one embodiment of a reactor system ofthe present invention.

FIG. 2 b is an exemplary view of another embodiment of the reactorsystem of the present invention

FIGS. 3 and 4 are charts showing the independent growth phases ofSynechococcus and Shewanella examples as compared to the binary culture.

FIGS. 5 a and 5b shows the efficiency of the use of lactate in thevarious tested cultures.

FIG. 6 shows the results of light testing of the Synechococcus culture.

FIGS. 7 and 8 show the quantity of electricity used for agitation andgas sparging per unit biomass.

DETAILED DESCRIPTION OF THE INVENTION

The following description includes the preferred best mode of oneembodiment of the present invention. It will be clear from thisdescription of the invention that the invention is not limited to theseillustrated embodiments but that the invention also includes a varietyof modifications and embodiments thereto. Therefore, the presentdescription should be seen as illustrative and not limiting. While theinvention is susceptible of various modifications and alternativeconstructions, it should be understood, that there is no intention tolimit the invention to the specific form disclosed, but, on thecontrary, the invention is to cover all modifications, alternativeconstructions, and equivalents falling within the spirit and scope ofthe invention as defined in the claims.

As a proof of principle, we have used a binary culture of aphotoautotrophic oxygenic cyanobacterium and a heterotrophic facultativeanaerobic bacterium and cultivated them in a non-aerated photobioreactorwith addition of organic carbon. During this process, the binary cultureproduced higher amounts of microalgal biomass without gas sparging (toremove O₂ produced during photosynthesis) or additional CO₂ injections.While specific examples are described hereafter and provided herein itis to be distinctly understood that the invention is not limited tothese described configurations but that a variety of additionalconfigurations and embodiments may be variously and alternativelyconfigured according to the particular needs and necessities of theuser. These binary culture systems of phototrophic organisms allow forefficient design and cost effective production processes for directingcarbon and nutrients flow from CO₂ and waste towards of biofuels:lipids, hydrocarbons and other preselected materials. The examples anddescriptions provided herein should therefore be seen as illustrative innature and not limiting.

To prove this concept various experiments were performed. In oneembodiment of the invention a binary culture of a photoautotrophicorganism Cyanothece sp. strain ATCC 51142 was cultured in associationwith a facultative anaerobic heterotrophic bacterium Shewanella sp.strain W3-18-1 on defined mineral medium which was not supplemented withCO₂ or bicarbonate salts as source of carbon. The mineral medium wassupplemented with lactate as source of carbon and reducing equivalentsto remove the O₂. During this process, both organisms in this binaryculture were dependent on the metabolism of the other: Shewanella sp.W3-18-1 consumed lactate and O₂ and produced CO₂ and Cyanothece sp. ATCC51142 incorporated the CO₂ into the biomass and produced O₂. During thisprocess both cultures in the photobioreactor grew without air spargingor addition of supplemental CO₂. In the presence of Shewanella sp.W3-18-1, Cyanothece sp. ATCC 51142 was able to grow at higher rates whencompared to single-organism culture under identical conditions withsparging with CO₂ and N₂.

In another embodiment of the invention an approach was utilized whereinbinary photoautotroph-heterotroph cultures were used to spatiallyseparate the processes of photosynthesis and photosynthate conversioninto useful products (FIG. 1). This in particular allows for thecultivation of readily-engineered heterotrophic strains for majorbiotechnology products using CO₂ and light instead of commodities suchas glucose, sucrose, or other agricultural or synthetic feedstocks.Particularly, phototrophic oxygenic microorganisms that have beengenetically or otherwise modified to produce and excrete a solubleorganic compound(s) such as glycerol, lactate, pyruvate, acetate or anyother organic compound that can be used by a heterotrophic organism asthe sole source of carbon and energy to grow and/or synthesize a productof interest. Moreover, O₂ as well as carbon and energy source(s) for theheterotrophic organism will be uniformly produced in the liquid cultureby phototrophic component of a binary culture, ensuring absence of shockby periodic excess or deficiency of nutrients and oxidants thatconventional types of cultivation usually suffer. An aerobic orfacultative anaerobic heterotrophic organism will consume O₂ produced asthe result of photosynthesis, thus dramatically decreasing mass transferenergy expenditure and simplifying photobioreactor design and operation.Additional CO₂ produced by heterotroph will be again recycled byphototroph thus ensuring efficient utilization of carbon source(s). Thebinary culture approach also allows the utilization of various carbonsources ranging from CO₂ from power plants to municipal wastes. Becauseof the robustness of the phototroph-heterotroph association, the binarycultivation provides a novel platform for the development ofconsolidated bio-processing methods leading to production ofcarbon-neutral products at reduced economic and energetic costs.

In one set of experiments one embodiment of the system, device andmethod of the present invention was compared against a prior art system,device and method. A description follows:

Materials and Methods:

Bioreactor:

A New Brunswick Scientific BioFlo 3000 bioreactor with a custom 7.5Liter vessel was used with a 5.5 Liter working volume. The pH was heldat 7.4 with 2 M NaOH and 2 M HCl when necessary. The temperature for allexperiments was 30° C. The high agitation experiments were done with 250rpm, and the low agitation with 50 rpm. Sparging was done with pure airat 2.8 L/min (optimal condition) for the Shewanella cultures, with 99%N₂ and 1% CO₂ for Synechococcus sp. PCC 7002 cultures, or with nobubbling for the Synechococcus 7002/Shewanella W3-18-1 co-cultures.Batches at 50 rpm agitation and 2.8 L/min aeration were also done withpure cultures of Synechococcus or Shewanella to show effects of lowmass-transfer on a pure culture (suboptimal condition 1). Two batches at50 rpm and 0.5 L/min aeration were done with Synechococcus (suboptimalcondition 2).

Photobioreactor:

A custom photobioreactor enclosure (developed by Pacific NorthwestNational Laboratory and shown in FIG. 2) was used for these experiments.This photobioreactor 10 includes a vessel 12 comprised of an outercylinder 14 made of black anodized aluminum and an inner cylinder 16made from borosilicate glass. The total height of the vessel 12 in thisembodiment was set out at 19.5 inches. The inner diameter was 5.274inches. The outer diameter (at the flange) was 6.5 inches. While thesedimensions were provided in this instance, it is to be distinctlyunderstood that the invention is not limited thereto. However, in thepresent embodiment it was found that a higher aspect ratio of height todiameter was more effective for enhancing growth within the chamber dueto the enhanced ability to allow light to penetrate into the center ofthe chamber. The bioreactor in this case had no additional ports placedupon the vessel 12. The anodized aluminum shell 14 acts as a mountingpoint for light emitting diodes (LED) 18 as a heat sink, and as a lightshield for ambient light. The black anodized coating of the aluminumabsorbs reflected light and acts as an efficient heat-transfer material.

A door 20 on the aluminum shell permits the user to view the innerworkings of the photobioreactor 10 as needed. Rubber fasteners 22securely close the door during normal operation to prevent light fromentering or exiting the photobioreactor. Foam rubber seals 24 and rubbergaskets 26 are used on mating surfaces of the photobioreactor 10 to keepit light-tight. The headplate 28 for the photobioreactor is configuredto allow probes (to monitor various factors such as pH, DO,level/foaming, and temperature) 30, tubing 32 and an impeller shaft 34for an agitator to reach the bottom of the vessel. (In this particularexample an additional port was added for a CO₂ probe for testingpurposes but such an addition is not required to allow for properfunctioning of the photobioreactor. Similarly an exhaust condenser wasincluded on this embodiment of the application (for testing purposes).In other applications various heat sinks 36 may be added. This is theonly photobioreactor design that combines a large working volume,thorough mixing in one common chamber, optimal light delivery, LEDlighting, selectable wavelength light, real-time light-intensitymeasurement and control, modeling of real outdoor lighting patterns andintensities, shielding from ambient room light, pH control, dissolvedoxygen control, temperature control, level or foam control, gas mixcontrol, off-gas monitoring, aseptic culturing conditions, andcontinuous-culture capability in one photobioreactor. This embodimentalso provides various advantages in regulating mixed or binary culturesto ensure that appropriate conditions are maintained to support desiredrates of growth and preserve and foster desired growth and/or materialconversions within said system.

Electronic circuitry 38 directs power from a power supply 40 to thebioreactor 10. A custom control software called Biolume (developed byDerek Hopkins of PNNL) interacts with sensors 40 within photobioreactorand the interactive electronic circuitry to regulate the desired levelsof light provided to the photobioreactor. For example in one embodimentof the invention the software controls maintains a specified lightpattern by coordinating the flow of electricity to light sources (LEDs)based upon input from sensors 40 and a preselected program includedwithin the device. This allows for light intensities to be varied overperiods of time so as to provide lighting cycles that best enhance thegrowth for materials that are included therein. For example, in oneembodiment of the invention light intensity and characteristics areadjusted over time to replicate the diurnal fluctuation in lightintensity which occurs in natural environments. Depending upon theindividual needs of the user any of a variety of characteristics may bevaried to achieve the desired results. This alterations and fluctuationmay include changes in intensity, wavelength, frequency, composition andother features as desired by a particular user.

To ensure that appropriate lighting conditions are maintained input fromthe sensors are correlated against a desired standard and a selfcalibrating program enables the light output to be appropriatelymoderated so as to ensure that an appropriate desired result isobtained. For example, in one embodiment of the invention input fromvarious sensors is compared to a desired output from a table of lightintensities that outline a specified pattern. If the receivedinformation is not aligned with the targeted amount, modification ismade so as to bring the light intensity within the system in line withthe targeted values. This feedback loop with a self-calibrating featurealso allows the electrical flow into the LEDs to be altered so as tocompensate for wear and degradation of the LEDs over time.

In the experiments described below, the light control system was tunedto deliver an identical current to each LED in the system. In thedemonstrated embodiment lighting for the photobioreactor is provided by16 extremely high output illuminators at 630 nm, and 16 at 680 nm. Eachilluminator contains 60 high efficiency InGaAlP diode chips (lightemitting diodes) made by Marubeni Corporation (Japan). A total of 1,920light emitting diodes are mounted to the inner wall of the aluminumshell. Each high output illuminator was positioned to be equidistant toadjacent illuminators. The cone of light emitted from each illuminatoroverlaps with adjacent illuminators to provide even lighting to thereactor surface. In other applications the wavelengths of the LEDs weremodified so that half were blue and half were red, the integration ofthe light control module with the sensors in that application allowedfor optimal growth conditions to be established and provided.

Six LI-COR Biosciences quantum sensors (for measuring photosyntheticallyactive radiation) were used to measure light intensities within thephotobioreactor. Three sensors measured incident light, and threemeasured transmitted light. The incident light sensors were mountedfacing the LEDs, while the transmitted light sensors were mounted facingthe center of the bioreactor. The light control software Biolume allowsProportional Integral Derivative (PID) control of incident ortransmitted light intensity. The lighting can respond to a manual setpoint, or automatically adjust power levels to the LEDs to maintain aset point. In one application light intensity measurements are made bycycling power to just the 630 nm LEDs, then to the 680 nm LEDs, then toboth 630 and 680 nm LEDs. Sampling duration and frequency can beadjusted by the user of BioLume. Control of light-intensity can be donefrom a large table of values allowing the user to reproduce “realoutdoor” lighting intensities and timing or create custom lightingschemes. Lights can be turned on, off, or intensity corrected at anytime. In the described application a 1 minute light sampling interval(with about 3 seconds total for light measurements) was demonstrated.PID control of light intensities allows the system to predict the futurebased on the past behavior.

The six quantum sensors were calibrated using a LI-250A Light MeterQuantum/Radiometer/Photometer made by LI-COR Biosciences. The referencelight sensor was randomly moved along the inner glass wall of thephotobioreactor for 15 seconds while the light meter averaged the lightintensities seen during the 15 seconds. The 15 second moving-averagelight intensities were plotted against the signal produced by the lightsensors at varying light intensities to produce a correlation plot. Theleast-squares best-fit equation was used to translate sensor-signal tolight-intensity in μEinsteins/m²/sec.

A+ medium was used to support organism growth for these experiments andsupplemented with lactate as needed. A+ medium contained the followingcomponents (concentrations in mM): Tris (8.255 mM), Na₂EDTA (0.0806 mM),KCl (8.0483 mM), CaCl₂.2H₂O (1.8120 mM), MgSO₄.7H₂O (20.2860 mM), KH2PO₄(0.3670 mM), NaCl (308.0082 mM), NH₄Cl (11.7540 mM-20.0 mM), Vitamin B12(2.95×10⁻⁶ mM), H₃BO₃ (0.5547 mM), MnCl₂.4H₂O (0.0218 mM), ZnCl₂ (0.0023mM), CoCl₂.6H₂O (0.00018 mM), Na₂MoO₄.2H₂O (0.00018 mM), CuSO₄.5H₂O(0.000012 mM). The pure Shewanella cultures were given 45 mM lactate andconsumed at most 27 mM of the lactate when grown to the highest density.Subsequent batches of the co-culture were given 25 mM lactate to avoidhaving excess lactate in the medium and were grown to lower biomassconcentrations as measured by optical density. No lactate was added forbatches of pure Synechococcus because preliminary experiments showedthat lactate neither was consumed by Synechococcus nor affected itsgrowth otherwise.

Shewanella W3-18-1 was grown in a 5.5 L batch at 250 rpm with 2.8 L/minsparging with air. Most of the culture was removed and replaced withfresh medium before growing the cells to the same final optical density.Reproducibility of the duplicate batches was shown before decreasing theagitation speed to 50 rpm. Growth rates and biomass yields were analyzedat the lower agitation speed.

Synechococcus sp. PCC 7002 was grown in batch phase for three replicatebatches at 250 rpm and 2.8 L/min sparging rate with 99% N₂ and 1% CO₂.Reproducibility of replicate cultures was evaluated and then theagitation was decreased to 50 rpm for replicate batches. The effects of50 rpm agitation on growth-rate and biomass yield were evaluated andthen the culture was allowed to grow to late log-phase for replicatebatches using 0.5 L/min bubbling. The late log-phase batches were doneto show that all other samples were considered mid-log phase. Thebiomass yield and growth-rate were determined and then the culture wasdiluted with fresh medium in preparation for co-culture growth withShewanella W3-18-1. Lactate was added to the medium to act as a sourceof carbon and energy for Shewanella and carbon for Synechococcus.

Shewanella W3-18-1 and Synechococcus 7002 were grown together inbatch-phase. Shewanella W3-18-1 used lactate to produce CO₂ needed bySynechococcus 7002. In turn, Synechococcus produced O₂ needed byShewanella W3-18-1 to oxidize lactate. After diluting the culture withfresh medium, the dissolved CO₂ concentration in the medium was very lowas measured by a dissolved CO₂ probe. To speed up growth of theco-culture on the 1^(st) batch after inoculation of both species, asmall amount of sodium bicarbonate was added (about 0.5 mM). Subsequentbatches of co-culture did not require supplementation with bicarbonate.

Results

Synechococcus grew at the same rate at 250 rpm and at 50 rpm agitationas long as 2.8 L/min gas-addition was used. However, the growth-ratedecreased slightly when 50 rpm agitation and only 0.5 L/min aeration wasused. Mass transfer through the high-aspect-ratio reactor (described inparagraph 23) was much more efficient than would be expected of a loweraspect ratio reactor with a shorter path-length for gas-exchange. Theimpact of mass transfer changes (agitation and aeration) have been muchmore obvious in a lower aspect ratio reactor as our previous experimentswith Shewanella showed. The maximum growth-rate of pure Synechococcuswas nearly the same as the co-culture of Synechococcus and Shewanella.This is because the co-culture is rate-limited by the growth ofSynechococcus as the result of both strains tight metabolic coupling.The lag phase of growth of Synechococcus was longer than the lag phasefor the co-culture (see FIG. 3), and Shewanella W3-18-1 grew faster thanthe co-culture or pure Synechococcus (see FIG. 4) as cyanobacteriagenerally has lower growth rates than aerobically grown heterotrophsunder optimal conditions. However, Shewanella did not use lactate asefficiently as the co-culture (see FIG. 5 a). The co-culture usedlactate 25% more efficiently than Shewanella alone because Synechococcusused CO₂ that was produced by pure Shewanella cultures. Analysis ofculture filtrates for organic acids revealed that lactate was not usedfully by coculture. Aacetate and sometimes formate (products of partiallactate oxidation by Shewanella) were found in the cocultureenvironment. When growth yield was calculated per mole of carbon used wefound that co-culture converts carbon to biomass 3.2 times better thanthe pure Shewanella culture (see FIG. 5 b).

Shewanella grew much better than expected at lower agitation (50 rpm).Again, this is due to the unusually high aspect ratio of the usedbioreactor. The 50 rpm culture did become O₂-limited as was indicated bythe red color of the culture due to the production and extracellularlocalization of cytochromes and accumulation of acetate. The biomassyield of the 50 rpm culture of Shewanella was much lower than the 250rpm culture by ash-free dry weight. Shewanella produced about 0.8 g/L ofash-free dry weight in a 12 hour growth period with 250 rpm (see FIG.3). This biomass concentration exceeded what was produced by theco-culture or Synechococcus in 30 hours (FIG. 3).

The co-culture of Shewanella and Synechococcus grew at about the samemaximum growth rate as pure Synechococcus (on an ash-free dry weightbasis). However, the co-culture used light 2.5 times more effectivelythan the pure Synechococcus culture (see FIG. 6). In other words, thebiomass yield as ash-free dry weight for a given amount of light was 2.5times as high for the co-culture as the pure Synechococcus culture.Obviously, the growth rate of the coculture was limited by growth rateof Synechococcus (see [0037]) as the result of tight metabolic couplingof two species.

The packed cell volume analysis showed that a co-culture of Shewanellaand Synechococcus had a stable ratio of about 1:1 by cell volume. Theamount of Synechococcus was always slightly higher that Shewanella, withSynechococcus to Shewanella ratios from 1.03:1 to 1.3:1. A packed cellvolume sample that was 43.45% (+/−0.76) Shewanella and 56.55% (+/−0.76)Synechococcus by volume was shown to have a percent by mass of 43.51%Shewanella and 56.49% Synechococcus. This means that the density of thecell pellets in g/L is equal for Synechococcus and Shewanella. We cantherefore relate the packed cell volume analysis directly to cell mass(ash free dry weight) by multiplying the fractional-share of thecell-volume by the combined ash-free dry weight.

The presence of products of incomplete lactate oxidation described inparagraph [0037] was the consequence of Shewanella growing faster thanSynechococcus and therefore it exhausted O₂ faster than Synechococcuscould produce it. As the result, Shewanella growth was limited by O₂.Under O₂ limitation Shewanella is known to convert part of lactate intoacetate and additionally can accumulate some formate; both thesecompounds cannot be used by Synechococcus, therefore decreasing itsgrowth and O₂ production rate. Hence, the process may become self-fadingunless some external O₂ or CO₂/bicarbonate is added. This conclusion issupported by the following experiments. Uncontrolled batches ofShewanella, Synechococcus, and binary co-cultures of these bacteria weregrown at room temperature (23-24° C.) in sterile Roux bottles (totalvolume 1 L) without gas sparging and mixing of cultural liquid. Bottleswere illuminated with cool white light at 55 μEinsteins/m²/sec. In thefirst set of experiments 0.6 L of A+ medium (supplemented with 9 mMlactate in case of pure Shewanella or binary cultures) was added,therefore leaving 0.4 l of air present in the headspace. Both purecultures did not produce significant growth for more than 250 hours,whereas binary culture fully used lactate for 120 hours. Biomass yieldof co-culture was 6 times higher than yield of pure Shewanella producedin controlled aerated bioreactor (see FIG. 3 a) and was 40 g of ash-freedry weight/mol of lactate carbon. Toward the end of active growth ofbinary culture most of the cells precipitated and formed a pellet on thebottom of the Roux bottle. This precipitation can be used as the way toconcentrate biomass in the process of growth by proper cultivationchambers design without necessity to centrifuge whole fermentationbroth, therefore potentially leading to significant additional energysavings on downstream processing. Additionally, packed cell volumeanalysis showed that co-culture of Synechococcus and Shewanella grownwith additional O₂ influx from the bottle headspace had a ratio 5:1 bycell volume, which means that Synechococcus made 5 times more biomassper lactate used that it did in binary culture bioreactor runs withoutexternal O₂ influx (see [0040]). In the second set of experiments theRoux bottles were filled with 960 ml of media. These experiments yieldedno growth for pure Synechococcus and Shewanella cultures; result forbinary culture was comparable with those obtained in photobioreactorexperiments. The overall binary culture growth limitation with O₂ causedby higher Shewanella growth rate and incomplete lactate oxidation can beovercome as follow. (1) Our previous experiments with Shewanella showedthat deletion of ackA gene encoding for acetate kinase abolishedincomplete lactate oxidation and acetate excretion under O₂ limitation.All lactate was converted into CO₂ and biomass, their ratio depended onthe level of O₂ supply. (2) Alternatively, addition of CO₂ orbicarbonate (see [0036]) will enhance specific rate of O₂ production bycyanobacteria in the initial stage of cultivation and as the resultincrease robustness of binary culture and final biomass production,yield, and degree of organic carbon source assimilation by the binaryculture.

The graph of electricity used (KWhrs) for illumination, sparging, andagitation per gram of ash-free dry weight (see FIG. 6) shows thatSynechococcus required the greatest investment for the smallest return;whereas, Shewanella produced the greatest amount of biomass for thesmallest input of electricity. This is because Shewanella did notrequire illumination, which was one of the costliest expenditures forelectricity in this experiment. Use of sunlight will significantlyreduce energy expenditures on binary culture illumination as well asoptimization of lighting conditions and overall process design. Theamount of electricity used for agitation and sparging per unit biomass(grams of ash-free dry weight) was highest for pure Synechococcus andlowest for the co-culture {57 times lower} (see FIG. 7). It should benoted that experiments conducted in Roux bottles used neither spargingnor mixing therefore potentially driving energy expenditures for thesepurposes almost to nothing. Since Shewanella required both bubbling andagitation, it required about 7 times more electricity per unit biomassas the co-culture. Thus, binary cultures not only provide advantage inrelation to efficiency because venting or sparging is no longer needed.There are also substantial cost improvements because there is no needfor aerating the mixtures and off-gas purification, the last absolutelynecessary in the industrial environments.

Unlike other methods described in the prior art the proposed approachaccomplishes CO₂ delivery/O₂ removal simultaneously with the process ofbiomass/product biosynthesis. This method of cultivation does not needsparging of a closed bioreactor with air or other gas mixture/vigorousmixing to deliver CO₂/remove produced O₂ from liquid medium. Thisimprovement leads to significant savings on energy consumption toaccomplish these tasks. It also allows designing photobioreactors of anydesired shape to ensure optimal illumination and space usage. Use ofhighly reduced organic compounds (for example wasted fats) in some caseswill help to consume externally added CO₂ or bicarbonate withoutnecessity to remove O₂ As source of organic matter many types of wastematerials can be used, for example biosludge produced from sewage watertreatment plants or starch solutions generated as the result ofindustrial potatoes processing that are typically wasted. Some of thesematerials may also be a cheap source of other nutrients such as N, P,and S sources.

In addition to the organisms previously described, a variety of otherorganism pairings are envisioned. These include pairs of Synechococcussp. PCC6038+Shewanella oneidensis MR-1 and Cyanothece ATCC51142+Shewanella sp. W3-18-1. Other possible candidates fromphototrophic side include microorganisms that are able to growautotrophically by using CO₂ as the source of carbon, light as thesource of energy, and water as source of electron, i.e. carry watersplitting and produce oxygen: Examples of such organism include but arenot limited to various types of cyanobacteria: nitrogen fixing, bothsingle cell (e.g. Cyanothece, some species belonging to Synechococcus)and filamentous (e.g. Trichodesmium, Anabaena, Nostoc), and non nitrogenfixing belonging to single cell (e.g. Synechocystis, some speciesbelonging to Synechococcus) and filamentous e.g. Arthrospira (formerSpirulina) as well as various genetically modified strains of theseorganisms. Other examples include microalgae: e.g. Haematococcuspluvialis, Clamydomonas etc., as well as various genetically modifiedstrains. Any heterotrophic obligatively aerobic or facultative anaerobicmicroorganism, belonging to archaea, bacteria, or eukaryotes, that areable to oxidize organic compounds to CO₂ using O₂ as electron acceptor,and use organic compounds for growth and/or biosynthesis of product ofinterest may also be utilized examples of such materials include: E.coli, Corynebacterium glutamicum, Saccharomyces cerevisiae and the like.

Another embodiment of a photobioreactor is shown in FIG. 2B. In thisembodiment the photobioreactor has several alterations and modificationsover the design previously discussed. First, this new design is madethrough an extruded process which allows for a more cost effectivedesign and allows for enhanced efficiencies related to both cost andfunction. In addition, this new design also includes integral heat sinksand ports for making the various lighting and harnesses for thelighting. This new design also offers space for twice as many LEDs asthe previous design because less space is used for the round heat-sinks.Cooling fans are automatically controlled by a temperature dependantprogrammable logic controller to increase the longevity of LEDs.

The present invention core forms an economically attractive way tocreate a variety of biobased products including but not limited tobiomass; Hz; organic fertilizers; biodiesel and biocrude oil; ethanol;amino acids; vitamins; antibiotics; polysaccharides and fine chemicals,for example D- and L-lactate as polylactates precursor. Thesemethodologies may also be utilized in a variety of other different waysincluding but not limited to biosludge and other organic wastesutilization in economically sound way. In addition other embodiments mayprovide other possibilities and potentials such as the ability toregulate CO₂ production/O₂ removal or CO₂ consumption/O₂ removal atdesired rates.

While various preferred embodiments of the invention are shown anddescribed, it is to be distinctly understood that this invention is notlimited thereto but may be variously embodied to practice within thescope of the following claims. From the foregoing description, it willbe apparent that various changes may be made without departing from thespirit and scope of the invention as defined by the following claims.

1. A closed bioprocessing system characterized by a container having anat least partially artificially lighted system operably connectedthereto
 2. The closed bioprocessing system of claim 1 wherein saidsystem is completely artificially lighted.
 3. The closed bioprocessingsystem of claim 1 wherein said closed system includes a chamber havingsensors positioned therein.
 4. The closed bioprocessing system of claim3 wherein said sensors are operatively interconnected to an artificiallighting system.
 5. The closed bioprocessing system of claim 1 furthercomprising a heat sink.
 6. The closed bioprocessing system of claim 1further comprising a mutually beneficial co-culture.
 7. The closedbioprocessing system of claim 1 wherein said cylinder has a height towidth aspect ratio of at least
 2. 8. The closed bioprocessing system ofclaim 1 wherein said cylinder further comprises an integrated heat sink.9. The closed bioprocessing system of claim 1 further comprising acontrol device that monitors and controls the quantity of light enteringinto said cylinder.
 10. A non-aerated photobioreactor for obtainingbiotechnology products using CO₂ and light together with a binaryculture made up of a combination of a phototrophic organism and aheterotrophic organism said photobioreactor having a chamber with aheight to diameter ration greater than two and lit by an artificiallighting system comprised of light emitting diodes (LEDs) arranged in aspaced configuration.
 11. The photobioreactor system of claim 10 whereinlight emitted from said LEDs is the only light within said system. 12.The photobioreactor system of claim 11 wherein said LED's are controlledby a system based upon inputs from sensors surrounding said chamber.